A fundamental question in biology involves the understanding of how metabolic pathways are modified in response to changes in substrate availability. Understanding how the partitioning of C1 carbon between oxidation and assimilation occurs after metabolic disturbance in the facultative methylotrophic bacterium Methylobacterium extorquens AM1 is the main focus of this research project. The hypothesis underlying this project is that the central enzyme methylene tetrahydrofolate (H4F) / tetrahydromethanopterin (H4MPT) dehydrogenase (MtdA) plays a dual role in methylotrophy, both in the production and consumption of the branchpoint intermediate formate, and as such, is a key regulatory element in balancing assimilatory and dissimilatory metabolism. The specific aims are twofold, 1) defining the role of MtdA in the H4MPT oxidative pathway and 2) defining the role of MtdA in the assimilatory flux redistribution that occurs when the cell experiences a metabolic perturbation. To study the role of MtdA in the H4MPT pathway, phenotypic studies of strains with different levels of expression relative to each other of the two methylene-H4MPT dehydrogenases (MtdA and MtdB) grown on methanol, compared to wild-type will be performed. We will assess methanol sensitivity, growth defects, and flux to CO2. Conditions affecting these parameters will be studied further by microarrays, activity assays, and metabolite analysis. These studies will test the hypothesis that MtdA is important in the production of formate in the H4MPT pathway and will provide information on how the cell responds when the normal balance is upset. The mechanism by which MtdA restricts flux to biomass will be assessed by first determining whether the effect occurs at the post-transcriptional or post-translational level. This level will be identified by measurements of enzyme and activity in appropriate growth conditions compared to transcripts. If a post-translational effect is found, biochemical and kinetic studies of purified enzymes in the presence of different effectors will be tested. Proteomics analysis will be performed to test for a covalent modification. Protein stability will be tested by pulse-labeling studies and mutation analysis will be used to assess translational control. Integration of these results into a metabolic model will be implemented and the relationship of this mechanism to the other roles of MtdA will be assessed, resulting in a greater understanding of how the cell dynamically regulates carbon flux through the formate branch point. The interplay of MtdA activity, nucleotides and other effectors, and post-transcriptional modification should illuminate a dynamic mechanism that poises the cell for efficient growth and transitions. Understanding this fundamental process has significance in two main ways. Because C1 metabolism is required for several biosynthetic reactions in all organisms, its understanding can be extrapolated to other systems. Addressing these questions will provide principles for understanding and manipulating microbes for applications involving new biotechnological and green chemistry techniques, bioremediation, and development of new therapeutics. PUBLIC HEALTH RELEVANCE: Understanding the regulation of carbon distribution between assimilation and oxidation will facilitate the manipulation of metabolic networks to be applied for societal benefit through biotechnology, bioremediation, green chemistry, and development of new therapeutics, using bacteria as chemical factories.